Human coagulation Factor X (FX), activated FX (FXa), and variants thereof, are used as therapeutic agents in blood coagulation disorders including, but not limited to, hemophilia and von Willebrand disease. FX, a vitamin K-dependent serine protease, is synthesized as a single chain precursor protein in the endoplasmic reticulum, with subsequent intracellular proteolytic furin cleavage in the Golgi apparatus before secretion by the producing cell into the blood stream, or into the culture medium in case of recombinant expression. Three furin cleavage sites in FX are responsible for proper FX proteolytic processing. The mature form of FX is a disulfide-linked two-chain molecule consisting of a heavy and light chain, formed after cleavage of the precursor protein. Further modifications of the molecule include γ-carboxylation of the light chain and N- and O-linked glycosylation of the activation peptide which is attached to the heavy chain.
Besides FX, further Vitamin K-dependent coagulation factors bearing the consensus recognition site Arg-X-Lys/Arg-Arg (SEQ ID NO:4) are substrates of the ubiquitously expressed endoprotease furin, also known as paired basic amino acid residue-cleaving enzyme (PACE). Adequate proteolytic processing of recombinant proteins of the coagulation cascade are impaired in cell culture expression systems due to intracellular processing limitations at high yield expression. Similar to von Willebrand Factor and coagulation Factor IX (FIX) which exhibit insufficient proteolytic processing at high expression rates in recombinant mammalian cells, FX secretion in low producing CHO cell clones is characterized by fully processed FX, whereas high producing clones comprise unprocessed single chain FX and multiple unprocessed forms of FX light chain, in addition to the correctly processed FX heavy and light chain species. Types and degrees of unprocessed FX light chain varied among individual cell clones and under different cell culture conditions such as cell density. Additional in vivo furin co-expression or post-cell culture in vitro furin incubation is needed to support the endogenous furin proteolytic machinery, facilitating intact protein cleavage.
Furin co-expression is indispensable for the expression of fully processed FX at high yield. However, to date no threshold level of furin has been reported that would ensure a high percentage of intact processed FX in cell culture systems. High levels of furin are toxic, therefore levels of furin expression by FX-producing mammalian expression systems must be balanced between levels that are toxic, yet potentially process 100% of the FX precursor protein, and those that are too low, resulting in healthy cell cultures in which suboptimal processed FX is produced.